U.S. patent number 3,583,230 [Application Number 04/736,360] was granted by the patent office on 1971-06-08 for sample injection method and apparatus.
This patent grant is currently assigned to Sondell Research Development Co.. Invention is credited to James A. Patterson.
United States Patent |
3,583,230 |
Patterson |
June 8, 1971 |
SAMPLE INJECTION METHOD AND APPARATUS
Abstract
Injector cartridges having filter material are placed in
receiving holes around the periphery of a rotating turntable after
a sample has been centrifuged into the filter material and the
sample is filtered during the centrifuging. The sample within the
cartridge is preeluted and then the turntable is rotated
sequentially positioning the cartridges beneath an extendable and
retractable loader which engages the cartridge. Desired zones of
the chromatographic spectrum of the preeluted sample are injected
through a capillary outlet in the cartridge into a chromatographic
system.
Inventors: |
Patterson; James A. (Los Altos,
CA) |
Assignee: |
Sondell Research Development
Co. (Palo Alto, CA)
|
Family
ID: |
24959579 |
Appl.
No.: |
04/736,360 |
Filed: |
June 12, 1968 |
Current U.S.
Class: |
73/864.85;
73/23.42; 210/198.2; 422/64; 436/177; 73/61.56; 210/361; 210/781;
422/70 |
Current CPC
Class: |
G01N
30/24 (20130101); G01N 30/6091 (20130101); Y10T
436/25375 (20150115) |
Current International
Class: |
G01N
30/00 (20060101); G01N 30/24 (20060101); G01N
30/60 (20060101); G01n 031/08 () |
Field of
Search: |
;73/421,421.5,422,422GC,423,424,425,425.2,425.4,421CA,23,23.1,53,61.1
;210/31C,198C,78,361,362 ;23/230,253 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Primary Examiner: Prince; Louis R.
Assistant Examiner: Post, III; Harry C.
Claims
I claim:
1. A sample injector assembly for automatically injecting samples
into a chromatographic system comprising: a plurality of sample
injector cartridges each having a channel therethrough containing a
chromatographic filter material, an inlet opening at one end of
said cartridge communicating with said channel for loading a sample
therein, and a capillary outlet opening at the other end of said
cartridge communicating with said channel ejecting a sample
therethrough; a turntable having a plurality of holes therethrough
from one side to the other around the periphery thereof adapted to
receive said injector cartridges from one side, and retain said
cartridges therein, each said hole including spring biasing means
to bias an injector cartridge inserted therein in the direction of
the side of the turntable from which said injector cartridge is
inserted; means for rotating said turntable; means stationary
relative to said rotating turntable positioned on one side at the
periphery thereof comprising an extendable and retractable loader
adapted to engage in sealing relationship the inlet opening of an
injector cartridge received in a hole in the periphery of the
turntable and extend in a direction against the biasing spring
means in said hole; a receiving base stationary relative to said
rotating turntable positioned on the side of the turntable opposite
and in alignment with the extendable and retractable loader, said
receiving base having an orifice therein adapted to receive the
capillary outlet opening of an injector cartridge in sealing
relationship upon engagement of a cartridge by said loader and
extension of said loader against a biasing spring means.
2. A method of preparing a sample and injecting the sample into a
chromatographic system comprising: centrifuging the sample into a
chromatographic filter material contained in an injector cartridge
and filtering the sample during centrifuging of the sample into the
injector cartridge; preeluting the sample in the chromatographic
filter material in the injector cartridge; positioning the injector
cartridge for injecting the sample into a chromatographic system;
and injecting desired zones of the chromatographic spectrum of the
sample from the injector cartridge into the chromatographic
system.
3. A method of preparing a sample and injecting the sample into a
chromatographic system comprising: centrifuging the sample through
a membrane filter into a chromatographic filter material contained
in an injector cartridge; centrifuging a buffer solution through
the membrane filter and into the chromatographic filter contained
in the injector cartridge; preeluting the sample in the
chromatographic filter material in the injector cartridge;
positioning the injector cartridge for injecting the sample into a
chromatographic system; injecting predetermined zones of the
chromatographic spectrum of the sample from the injector cartridge
into the chromatographic system.
4. A sample injector assembly for automatically injecting samples
into a chromatographic system comprising: a fluid line arranged for
directing fluid and sample material to said chromatographic system;
a plurality of sample injector cartridges, each having a chamber
therein, chromatographic filter material located in said chamber,
means defining an inlet into said chamber for loading a sample
therein, and means defining an outlet from said chamber for
ejecting a sample therefrom; table means having a plurality of
holes formed therethrough to receive the injector cartridges, each
said hole including resilient biasing means to bias an injector
cartridge inserted therein in a direction away from the table; said
fluid line connected at one end to said chromatographic system and
at its other end to a position adjacent said table means; means
translating said table means relative to the fluid line for
successively positioning said holes in alignment therewith; and
means engaging an injector cartridge contained within a hole in the
table in alignment wit said fluid line to depress the injector
cartridge against the resilient biasing means, thereby to engage
the outlet from said injector cartridge and the end of the fluid
passageway in sealing relationship.
5. A sample injector for a chromatographic system comprising: an
elongated injector cartridge having a channel therethrough
containing a chromatographic filter material, an inlet opening at
one end of said cartridge communicating with said channel for
loading a sample therein and a capillary outlet opening at the
other end of said cartridge communicating with said channel for
ejecting a sample therethrough, a membrane filter in the inlet
opening end of said cartridge, and at least one screen placed in
the inlet opening end of said injector cartridge to prevent
pressure buildup in the injector cartridge on the chromatographic
filter material.
6. A sample injector for a chromatographic system comprising: an
elongated injector cartridge having a channel therethrough
containing a chromatographic filter material, an inlet opening at
one end of said cartridge communicating with said channel for
loading a sample therein and a capillary outlet opening at the
other end of said cartridge communicating with said channel for
ejecting a sample therethrough, said chromatographic filter
material comprising a plurality of microspherical cross-linked
polymer resin beads, and a tubular support column coaxially mounted
within the channel and fine mesh screening fixed at each end of the
support column for containing said resin beads.
Description
This invention relates to a new and improved method and apparatus
for preparing and injecting samples into a chromatographic system
particularly applicable for injecting samples of animal fluids for
chromatographic analysis of amino acids.
According to present chromatographic methods, liquid samples are
supplied directly to a chromatographic column by manual or
automatic injection. Because the sample is introduced directly, the
column must be regenerated between samples to remove impurities. In
addition, high pressures accompanying the injection of the sample
can compress the absorptive material used in the column and thereby
distort the resulting chromatogram of the sample. Another problem
encountered in practicing present techniques, is that in the
sequential steps performed for injecting sequential samples air can
be introduced into the operating column further distorting the
chromatogram.
The object of the present invention is to provide a method and
apparatus wherein a plurality of samples can be automatically and
sequentially injected into a chromatographic system for
analysis.
According to one aspect of the present invention, a sample injector
is provided in which large molecules such as proteins first removed
by centrifuging the sample through a membrane filter and into an
injector cartridge containing chromatographic filter material. The
sample is then preeluted in the carriage so that predetermined
zones of the chromatographic spectrum of the sample can be retained
in the cartridge or injected into a chromatographic system. A
sequence of samples can thus be continuously applied to a
chromatographic column without the necessity of column regeneration
between samples.
In accordance with another aspect of this invention the samples
applied to the chromatography column are changed continuously and
automatically with a positive displacement of all air so that air
is not introduced into the operating column. The injector cartridge
also prevents a pressure buildup in the sample and buffer which
might be transmitted to the chromatographic column. The sample
injector is particularly suitable for the chromatographic analysis
of animal fluids, and predetermined amino acids or other molecules
can be retained in the injector cartridge and only selected amino
acids from the sample injected into the chromatographic column. The
chromatographic column can thus be used continuously without
regeneration and only the injector cartridges need be regenerated.
The invention is also intended to provide means for automatically
injecting consecutive samples into a chromatographic system.
In order to accomplish these results, the present invention
contemplates providing a plurality of injector cartridges each
having a channel therethrough containing a chromatographic filter
material, an inlet opening at one end of the cartridge
communicating with the channel for loading a sample, and a
capillary outlet opening at the other end of the cartridge
communicating with the channel for retaining a sample therein until
it is ejected through the capillary outlet.
According to another aspect of the invention, a turntable is
provided having a plurality of holes through the turntable from one
side to the other around the periphery thereof adapted to receive
and retain the injector cartridges therein, each hole including
spring biasing means to bias an injector cartridge inserted therein
in the direction of the side of the turntable from which the
injector is inserted.
Samples to be analyzed in a chromatographic system are first
centrifuged into respective injector cartridges through loading
columns and membrane filters to remove protein. A buffer solution
is also introduced into the cartridge by centrifuging and each
cartridge is then placed in the turntable. The sample in each
injector cartridge is preeluted for a predetermined period and
positioned by the turntable beneath an extendable and retractable
loader adapted to engage in sealing relationship the opening of the
injector cartridge and extend in a direction against the biasing
spring in the hole of the turntable in which the cartridge is
seated. The injector cartridge is then pushed by the extending
loader into an orifice in a receiving base positioned on the other
side of the turntable from the loader for injection of
predetermined zones of the chromatographic spectrum of the sample
into a chromatographic system.
From the time that the sample is centrifuged into the injector
cartridge and the buffer introduced over the sample, the sample is
isolated from the air. Before the extendable loader engages an
injector cartridge held in the turntable, a droplet of buffer falls
on the inlet opening of the cartridge so that air is not introduced
upon sealing engagement of the inlet opening by the extendable
loader. Back pressure in the orifice of the receiving base from a
chromatographic system also backs up the buffer level into the
orifice to meet the capillary outlet from the injector cartridge.
No air is thereby introduced into the system during the sample
injection. The capillary outlet also serves to prevent premature
flow-out of the sample and buffer.
The chromatographic filter material provided in the injector
cartridge can be a plurality of cross-linked polymer resin beads
mechanically supported within a channel through the injector
cartridge. The resin beads can be mechanically supported by screens
at either end of the cartridge which also serve to prevent pressure
buildup on the resin beads.
Other objects, features and advantages of the present invention
will become apparent in the following specification and
accompanying drawings.
In the drawings:
FIG. 1 is a side cross-sectional view of an injector cartridge
embodying the present invention.
FIGS. 1A and 1B are plan views of two types of caps provided over
the outlet from the sample injector.
FIG. 2 is a plan view from the top of the injector cartridge
illustrated in FIG. 1.
FIG. 3 is a side cross-sectional view of an injector cartridge and
loading column in sealing engagement within a centrifuge tube.
FIG. 4 is a fragmentary plan view of an automatic sample injector
embodying the present invention.
FIGS. 5 and 6 are detailed side views of the extendable and
retractable loader for the automatic sample injector showing an
injector cartridge in the turntable before and during injection of
a sample respectively.
As broadly set forth above, the present invention provides a method
and apparatus for automatically injecting a plurality of samples
into a chromatographic apparatus wherein separate samples are
inserted into sample injectors containing a chromatographic filter
material and these sample injectors sequentially inserted in the
stream of the chromatographic apparatus.
In the embodiment of the present invention illustrated in FIG. 1
there is provided a sample injector 10 having an elongated housing
11 of plastic or other suitable material with a chamber or
elongated channel formed centrally therein. The channel 11 is lined
with a tubular support column 12 of glass or suitable plastic
material for supporting the chromatographic absorptive filter
material 13 such as microspherical resin beads. Microspherical
resin beads for use as the filter material can be formed of the
cross-linked copolymer such as, for example, a styrene and divinyl
benzene copolymer as set forth in more detail in my U.S. Pat.
application Ser. No. 530,051, now Pat. No. 3,463,320, entitled
"Microsphere Filter and Method of Filtration" filed on Feb. 25,
1966. The resin beads 13 are mechanically retained within the
support column 12 at each end by screens 14 such a 10 micron mesh
screening.
At one end of the sample injector cartridges there is provided an
inlet opening 15 communicating with the central channel in which
the chromatographic filter material is retained. Positioned at the
inlet opening of the cartridge over the screen 14 is a membrane
filter 16 sandwiched between glass mat filters 17 which support the
membrane filter and provide a prefilter for liquid samples entering
the cartridge inlet. At the opposite end of the injector cartridge
from the inlet 15, the cartridge is provided with a tapered
truncated conical projection 18 having a capillary outlet 19 formed
therein communicating with the elongated channel 11' in the
cartridge containing the chromatographic filter material 13. The
capillary outlet 19 serves to retain fluid in the cartridge before
injection. A cap 19a of teflon or other suitable material may be
provided over the capillary outlet 19 and truncated conical
projection 18, having an angled-slit cut through the cap as
illustrated in FIG. 1 and FIG. 1A to further retain fluid and
release fluid from the cartridge only upon application of pressure
on the fluid in the cartridge. Cross angled-slits may also be
provided in the cap as illustrated in the cap 19b of FIG. 1B. The
injector cartridge 10 illustrated in FIG. 2 is circular in cross
section and is provided with a flat surface 20 on one side thereof
to facilitate use in the automatic sample injector as hereinafter
described.
In accordance with one aspect of the present invention a liquid
sample is introduced into the sample injector cartridge 10 by
centrifuging the sample through the membrane filter 16 into the
column of resin beads or other chromatographic filter material 13.
To this end, a loading column 30 is provide as illustrated in FIG.
3. The loading column is formed of an elongated tube of plastic or
other suitable material having an inlet 31 for inserting the sample
and an annular projection 32 at the opposite end supporting an
O-ring seal 33 for engaging the inlet opening 15 of the injector
cartridge 10 in sealing relationship. The samples may then be
centrifuged through an outlet passageway 34 through the annular
projection 33 of the loading column 30 into the injector cartridge
10.
According to the operation by one aspect of this invention, the
injector cartridge 10 is first washed with a buffer solution such
as a citrate buffer solution of pH 2.2, and 0.2N in sodium ion, by
centrifuging the injector cartridge 10 alone in a centrifuge tube
35. The washings at the bottom of the centrifuge test tube 35 are
discarded. The liquid sample such as an animal fluid or a serum
with adjusted pH is then applied to the loading column 30 which has
been inserted into the inlet opening 15 of the injector cartridge
10. The liquid sample is then centrifuged into the cartridge
through the membrane filter 16 including the glass mat filters 17
and screen 14. Centrifuging is accomplished in a centrifuge test
tube 35 as illustrated in FIG. 3. To insure that the liquid sample
has been completely transferred to the injector cartridge, a buffer
of the same pH as the sample may be introduced into the loading
column 30 and washed through the system by centrifuging in the same
manner. The screens 14 which mechanically support the resin beads
during loading of the sample into the injector cartridge.
Sufficient buffer is applied during sample loading into the
injector cartridge so that the liquid level after centrifuging does
not fall below the level of the injector cartridge in the
centrifuge tube.
The loaded injector cartridge and loading column are then removed
from the centrifuge test tube and the loading column separated from
the cartridge. After removing the loading column 30, the glass mats
17 and membrane filter 16 are also removed from the top of the
injector cartridge leaving the top screen 14 in place. Proteins
filtered by the membrane filter are thereby removed eliminating a
source of contamination. The injector cartridge 10 is then inserted
into the automatic sample injector assembly illustrated in FIGS. 4
and 5 for injecting the sample into a chromatographic system.
The automatic sample injector assembly includes a drum or turntable
40 having a plurality of holes 41 formed therethrough from one side
to the other around the periphery of the turntable. A flat disc 42
is provided centrally on the turntable 40 so that it slightly
overlaps the edge of each of the holes 41 formed around the
periphery of the turntable 40. Each hole 41 is adapted to receive a
sample injector cartridge 10 with the flat edge 20 of the cartridge
facing the disc 42 so that it will fit into the hole 41. The
cartridge 10 is then rotated so that it is retained in the hole 41
by the disc 42 until rotated with the flat edge 20 again facing the
disc 42. Each hole 41 includes a biasing spring 43 coaxially
positioned therein and adapted to engage an annular projection
around an injector cartridge to spring bias an inserted injector
cartridge against the bottom of disc 42.
Means are provided for rotating the turntable 40 relative to an
extendable and retractable loader 45 positioned at the edge of the
turntable. The extendable and retractable loader 45 includes an
extendable and retractable annular projection or piston 46
positioned over the turntable and in alignment with the inlet
opening 15 of injector cartridges 10 as they are sequentially
positioned beneath the loader 45 by rotation of the turntable 40.
The piston 46 on loader 45 extends and retracts in a direction
perpendicular to the face of the turntable 40 and supports an
O-ring seal 47 therearound. Upon extension of the piston 46, the
piston enters and engages the inlet opening 15 of a cartridge 10 in
sealing relationship and pushes the cartridge downward in a
direction against the biasing spring 43. On the lower side of
turntable 40, i.e., the side opposite the extendable and
retractable piston 46, there is provided a receiving base 50
stationary relative to the rotating turntable 40. The receiving
base 50 includes an orifice 51 and O-ring seal 51a adapted to
receive the truncated conical extension 18 at the outlet end of an
injector cartridge in sealing relationship as illustrated in FIG.
6. The orifice 51 terminates in a passageway 52 connected to a tube
53 which leads to a chromatographic system in which the sample is
to be injected. Upon extension of the piston 46 from the loader 45
the capillary outlet end of the cartridge 10 is inserted into the
orifice 51 so that a buffer under pressure in tube 54 may be
transferred through the piston 46 and injector cartridge to inject
the sample through the tube 53 into a chromatographic system.
After a loaded injector cartridge is placed in the turntable 40,
the cartridge may be delivered to the stationary loader 45
according to a controlled timing sequence. The controlled timing
sequence of delivery of cartridges by the turntable 40 may include
a predetermined preelution cycle prior to positioning the cartridge
into position beneath the loader 45 for injecting the sample into a
chromatographic system. By preelution of the liquid sample in the
cartridge, amino acids or other molecules may be removed from the
sample to a controlled initial level in the chromatographic
spectrum of the liquid sample. The cartridges are removed from the
injection position beneath the loader 45 after a predetermined time
interval so that amino acids or other molecules beyond a desired
level in the chromatographic are retained in the cartridge. After
injection of predetermined zones of the chromatographic spectrum of
the sample into a chromatographic system, the cartridges are
removed from the injection position by turntable 40 for
regeneration of the filter column of resin beads contained in the
cartridge in preparation for loading another liquid sample into the
cartridge by centrifuging as heretofore described.
Once a liquid sample and buffer have been centrifuged into the
injector cartridge, the sample is maintained in buffer solution and
isolated from air throughout the subsequent stages of sample
delivery. When the sample injector is removed from the centrifuge
test tube after centrifuging, flow-out from the cartridges is
prevented by the capillary outlet 19 and cap 19a. The cartridge is
then inserted in one of the holes in the periphery of the turntable
40. Before each cartridge is delivered to the loader 45 and
extendable piston 46 for insertion of the end of the cartridge into
the orifice 51 of the receiving base 50, back pressure from the
chromatographic system through tube 53 causes the level of buffer
to rise in the tapered orifice 51 to receive the capillary outlet
19 and tapered end 18 of the cartridge so that air is not
introduced into the system. Furthermore, as a cartridge is
delivered by the turntable 40 into alignment beneath the piston 46
on the loader 45, a droplet of buffer falls from the piston 46 onto
the top of the cartridge in the inlet opening 15 so that when the
piston 46 engages the opening of the injector cartridge forcing the
tapered end 16 of the cartridge into the orifice 51, an excess of
buffer occupies the space in the inlet so that again air is not
introduced into the system. The excess buffer is forced out and
collected in a waste tube as the piston 46 extends into the
cartridge 10. After the sample is injected from a cartridge into a
chromatographic system, the turntable rotates and the cartridge may
then be processed through a regeneration cycle. The cartridge is
then removed by rotating the cartridge until the flat edge 20 faces
the disc 42 so that the biasing spring 43 ejects the cartridge for
loading another sample by centrifuging.
Although only certain embodiments of the present invention have
been shown and described, other adaptations and modifications would
be apparent without departing from the true spirit and scope of the
invention.
* * * * *